Calibrating the thermal cyclers is an important process to take care of them. That is why all points of view must be clear to calibrate one of them. Among these are molecular biological points of view, which focus on copying a PCR reaction to the view of metrologists.
That is why the calibration must be carried out by qualified personnel, that is, no ordinary person can try it. Remember that years of study are carried out for this. The views should also be carried out under defined environmental conditions that allow the process, as well as the circumstances.
Calibration by imitation of the PCR process
If we take it from the perspective of a molecular biologist, this is the best way to calibrate any thermocycler as long as it is the process that mimics the PCR process. The correct process is to put tuvos in block wells, fill the reaction mixture and place the sensors. Then proceed to close tubes and thermal cyclers with the thermal cover in order to develop the PCR protocol used in the laboratory.
This whole process must be under a real temperature in a tube filled with a particular reaction mixture. All this must happen during the PCR protocol carried out from a particular instrument.
Each mix combination, had or until the protocol and calibration instrument should be repeated. This makes the workload much greater. That is why the results cannot even be compared between them. Not even to any kind of norm or specification established by the manufacturer.
Keep in mind that this way of calibrating would literally collect many variables out of control. Therefore, adding a large component of uncertainty is the best way to end with uncertainties that range in the range of more than 2 ° C. Now, when the uncertainty is about 2 º C, and at the same time the measurement of 96 º C, it happens that the temperature of the thermal cycler can occur in any range between 94 º C and 98 º C.
It should also be borne in mind that the temperature difference could have repercussions on the inactivation of Taq polymerase. Therefore, the level of uncertainty is not at all pleasant in the calibration of the thermal cycler. However, if the PCR process is copied it may seem like a tempting option.
Representative process calibration
From a perspective, the best way to calibrate a utensil such as the thermal cycler is by representative and standard measurement of the PCR process. The method allows the calibration of all standard PCR. The dynamics and its static part must be taken into account during the PCR process. This is important since there are problems that cannot be known with the naked eye, or rather with the static control of a PCR.
Temperatures close to denaturation, elongation and hybridization should be evaluated one by one. The effects they may have may vary depending on the temperature.
Calibration according to international standards
If you work in the meteorology area, this could be the perfect area to calibrate a thermal cycler. In the matter of meteorologist, a calibration is defined as necessary operations under some specific conditions.
During this process traceability is guaranteed by making use of reference standards and expression of values that are responsible for calibrating in SI units. However, also because of that uncertainty calculated from the calibration result. In addition to the fact that the same calibrations are only made by engineers specialized in calibration who are literally competent and qualified to perform such action.
How to know if a calibration is completely safe
- Calibration must be measured with thermal characteristics that include a PCR result. Among them the accuracy, excess or reach, the cool heat speed, retention time and uniformity.
- A dynamic process must be carried out instead of a static one.
- It is essential to perform through several channels to exclude time effects between wells.
- It is very important to have controlled environmental conditions.
- Authorized personnel must have the necessary experience to carry out these actions.